The introduction of biologically active molecules, for example DNA, RNA or proteins, into living cells may, e.g., serve to examine the biological functions of these molecules and is, moreover, an essential precondition for the success of the therapeutic use of these molecules, e.g., in gene therapy. A preferred method for introducing external molecules into the cells is called electroporation, which unlike chemical methods limits undesirable changes in the structure and function of the target cell. In electroporation the external molecules are introduced into the cells from an aqueous solution, preferably a buffer solution specifically adapted to the cells, or a cell culture medium, via a short current flow, i.e., e.g., the pulse of a discharging capacitor which renders the cell membrane permeable to the external molecules. The temporary “pores” that are formed in the cell membrane allow the biologically active molecules to first reach the cytoplasm in which they may already perform their function or exert any therapeutic action to be examined, and then, under certain conditions, to also reach the cell nucleus as it is required, e.g., in gene therapy applications. Due to the short application of a strong electrical field, i.e. a short pulse with a high current density, cells, cell derivatives, sub-cellular particles and/or vesicles may also be fused. In this so-called electrofusion the cells are, e.g., initially brought into close membrane contact by an inhomogeneous electrical alternating field. The subsequent application of an electrical field pulse leads to interaction between membrane parts, which ultimately results in fusion. Devices comparable to those used for electroporation may be used for electrofusion.
Smaller volumes are generally treated in a batch process in relatively simple vessels. The solution or cell suspension, respectively, is frequently located in a cuvette, i.e. a narrow vessel open at the top, which in the vicinity of the bottom has two opposing, parallel electrodes in the lateral walls which serve to apply the electrical voltage. However, such vessels are unsuitable for treating larger volumes as the reaction space available for the electrical treatment is limited by the limited maximal distance between the electrodes. Thus, flow-through processes in which the cell or vesicle suspension is continuously or discontinuously fed through the reaction space between the electrodes are preferred for the electroporation or electrofusion of larger volumes.
U.S. Pat. No. 6,150,148, for example, discloses a cuvette modified for flow-through processes. The port of the cuvette is sealed by a cap through which a feed line is guided. At the bottom in a region between the electrodes the cuvette has an additional port to which a discharge is connected. Because of this arrangement the suspension to be treated can be fed through the feed line into the reaction space and exit it through the discharge. Due to repeated, continuous or discontinuous exchange of the suspension in the reaction room and the respective repeated pulsing, larger volumes can be treated with this cuvette.
U.S. Pat. No. 6,150,148 also discloses flow-through chambers which are of tubular or slotted design and at their ends each have a connection for an input and an output channel. The chambers themselves represent an oblong reaction space which is enclosed by two cylindrical, concentrically arranged or flat electrodes having plane-parallel configuration. These devices also allow larger volumes to be treated by repeated pulsing as they are fed through the chamber.
All references mentioned herein are incorporated herein by reference in their entirety.
During electroporation or electrofusion in flow-through processes the formation of gas bubbles by electrolysis poses, next to heating of the suspension, a significant problem. The very high currents that are often required for these processes leads to large numbers of small gas bubbles that are formed by electrochemical processes in the electrolyte solution in which the cells or vesicles to be treated are suspended. These bubbles disturb the flow of the suspension through the chamber and may result in a backflow of the suspension already treated into the chamber. This, on the one hand, leads to results that are no longer reproducible and on the other hand, if living cells are treated, to an increased mortality rate.
Thus, there is a need for a device and a method for flow-through electroporation or electrofusion in which a directed flow of the fluid to be treated through the chamber or reaction zone is guaranteed and a backflow of the fluid already treated into the chamber or the reaction zone, respectively can be avoided.